Primary hepatocellular carcinoma is a malignant disease that occurs at a high frequency in various countries over the world. As a result of large outbreaks of hepatitis B and C over the world, the incidence of hepatocellular carcinoma has been rapidly increasing in Asian and European countries. Taking into consideration the long incubation period from infection with hepatitis virus to the onset of the disease, it is anticipated that such tendency will continue over the coming fifty years. Hepatocellular carcinoma whose condition has become worse has a poor prognosis. Thus, it is desired to rapidly develop a new treatment strategy.
On the other hand, with the development of molecular biology and tumor immunology in recent years, it has been revealed that cytotoxic (killer) T cells and helper T cells recognize peptides formed by decomposition of proteins highly expressed specifically in cancer cells, which are presented on the surfaces of the cancer cells or antigen-presenting cells via HLA molecules, and that they exhibit an immune reaction for destroying such cancer cells. Moreover, a large number of tumor antigenic proteins and peptides, which stimulate such an immune reaction to attack cancers, have been identified, and clinical application of an antigen-specific tumor immunotherapy has been advanced.
HLA class I molecules are expressed on the surfaces of all nuclear cells in a body. Peptides derived from decomposed cytoplasmic and nuclear proteins are bound to HLA class I molecules, and they are expressed on the surfaces of such cells. On the surfaces of normal cells, peptides derived from normal autologous proteins bind to HLA class I molecules, and T cells of the immune system neither recognize nor respond to such peptides bound to HLA class I molecules. On the other hand, in a process in which normal cells are converted to a cancer, such cancer cells may express large amounts of proteins, which are hardly expressed or are only expressed in small amounts in normal cells. If a peptide generated by decomposition in the cytoplasm of such a protein that is highly and specifically expressed in a cancer cell binds to an HLA class I molecule and is expressed on the surface of such a cancer cell, a killer T cell recognizes the peptide and destroys only the cancer cell. In addition, by administering such a cancer-specific antigen or peptide to an individual body, it is possible to destroy cancer cells and suppress the growth of a cancer, without impairing normal cells. This is referred to as cancer immunotherapy using a cancer-specific antigen. Moreover, an HLA class II molecule is mainly expressed on the surface of an antigen-presenting cell. Such an HLA class II molecule binds to a peptide derived from a cancer-specific antigen generated by incorporating the cancer-specific antigen from outside the cell and decomposing it in the cell, and it is expressed on the surface of the cell. A helper T cell, which has recognized the peptide bound by HLA class II molecule, is activated to generate various cytokines that activate other immunocompetent cells, so as to induce or reinforce an immune reaction against a tumor.
Thus, if an immunotherapy targeting an antigen that is highly and specifically expressed in such a cancer can be developed, it can become a treatment method for effectively eliminating the cancer alone, without impairing normal autologous organs. Moreover, it is anticipated that such an immunotherapy can become a treatment method applicable to patients suffering from a terminal-stage cancer, for whom no other treatments can be implemented. Furthermore, if a cancer-specific antigen and peptide are administered in the form of a vaccine to a human having a high risk of developing such a cancer, there is a possibility that the onset of the cancer can be prevented.
It has been reported that, in normal tissues, an α-fetoprotein (AFP) is expressed only in the prenatal period, and that it is what is called a carcinoembryonic protein whose expression is activated again in many hepatocellular carcinomas. In addition, several types of mouse and human T cells recognize a peptide epitope derived from APP presented by an MHC class I molecule. During the developmental stage, a fetus is exposed to AFP existing at a high level in plasma. However, mature T cells do not acquire complete immunological tolerance to AFP, and AFP-specific T cells are detected in peripheral blood. That is to say, such a carcinoembryonic protein can be a target of immunotherapy.
There are various methods for treating hepatocellular carcinoma. However, the prognosis of such hepatocellular carcinoma is worse than those of other types of cancers, and thus this cancer is considered to be an intractable cancer. This may be because hepatocellular carcinoma develops on the basis of liver cirrhosis and thus patients with hepatocellular carcinoma have poor hepatic functions. This may also be because although a mass of cancer has been treated, another cancer develops from another site. Accordingly, it has been necessary to rapidly develop a novel treatment strategy. If an immunotherapy targeting an antigen that is highly and specifically expressed in hepatocellular carcinoma can be developed, there is a possibility that such an immunotherapy will become a therapeutic method for effectively eliminating cancer alone without impairing normal autologous organs. Moreover, it is anticipated that the aforementioned immunotherapy can be a therapeutic method, which is available for patients who are in terminal stage of cancer, and further, for patients whose hepatic functions are too poor to allow other treatments to be carried out. At present, it is said that, in Japan, more than 2,000,000 people are infected with hepatitis C virus, and that such people are, potential hepatocellular carcinoma patients. There is a possibility that the aforementioned immunotherapy will be also applied to prevent such infected patients from actually being suffering from hepatocellular carcinoma.
Melanoma is one type of skin cancer, which is often called malignant melanoma. There are many types of skin cancers. Among such skin cancers, melanoma is classified as having the highest grade of malignancy, and thus it is greatly feared. Among cells that constitute skin, several cells generate melanin pigment. Such cells are called melanocytes. When such melanocytes become cancerous, melanoma occurs.
In Japan, the incidence of melanoma varies from 1.5 to 2 people in 100,000 in the general population. Thus, it is estimated that approximately 1,500 to 2,000 people develop melanoma per year. On the other hand, in the Western countries, more than a dozen of people develop melanoma in 100,000 in the general population. In particular, in Australia, twenty or more people develop such melanoma in 100,000 in the general population, and thus it is said that the incidence of melanoma in Australia is the highest in the world. Under such circumstances, people who live in Europe, the United States, and Australia are interested in melanoma, and they pay attention to the occurrence of melanoma. In addition, the frequency of occurrence of melanoma has been increasing, particularly among Caucasians, as a result of an increase in exposure to ultraviolet rays due to a reduction in the ozone layer in the atmosphere caused by environmental destruction. Moreover, the occurrence of melanoma tends to be increasing year after year in Japan as well. According to recent studies, the annual death toll from melanoma in Japan has increased to approximately 450. Melanoma develops regardless of age. However, the incidence of this disease increases for those over 40, and it is the highest for those in their 60's and 70's. The onset of this disease in childhood is extremely rare, but this does not mean that the disease never develops in childhood. Recently, the occurrence of melanoma tends to be increasing in young patients in their 20's and 30's. Melanoma develops regardless of sex, and both male and female patients suffer from this disease. In the case of Japanese patients, the site at which melanoma is most likely to develop is the sole (the sole of the foot), and it accounts for 30% of all cases of melanoma. As characteristics of Japanese patients, melanoma also develops in the foot and the nail portions of the fingers. In addition, as in the case of Western patients, melanoma develops in all parts of the skin, such as the body, hand, foot, face, and head, among Japanese patients as well.
First, the present inventors have performed genome-wide gene expression analysis, including regarding 23,040 kinds of human genes, utilizing cDNA microarray analysis. The inventors have analyzed expression profiles of these genes in 20 cases of primary hepatocellular carcinomas and in various types of normal organs including those present in the prenatal period. As a result, the inventors have found that glypican-3 (GPC3) is expressed in the liver, kidney, and lung during the prenatal period, and also that such glypican-3 is highly expressed in many hepatocellular carcinomas, although it is hardly ever expressed in normal adult organs, although it is expressed in placenta. The inventors have further reported that such GPC3 is a secretory protein, that such GPC3 can be detected in the serum of 40% of hepatocellular carcinoma patients by the ELISA method, and that this is useful as a novel tumor marker of hepatocellular carcinoma (Nakatsura, T. et al., Biochem. Biophys. Res. Commun. 306, 16-25 (2003)). Moreover, they have also reported that GPC3 is detected in the serum of melanoma patients, and that it is also useful as a tumor marker of melanoma (Nakatsura, T. et al., Clin. Cancer Res. 10: 6612-6621 (2004)).
The present inventors have already identified a GPC3 peptide, which binds to HLA-A24 and is presented to a human killer T cell, and which is useful for an immunotherapy that targets patients with HLA-A24 positive hepatocellular carcinoma or melanoma. The inventors have carried out an animal experiment using BALB/C mice that express mouse Kd molecules, to which a peptide having the same structure as a peptide binding to HLA-A24 binds. Through this, they have demonstrated the effectiveness of immunotherapy using the aforementioned peptide and have already reported the results (International Application No. PCT/JP2004/016374; International Filing Date: Oct. 28, 2004). In normal organs, since GPC3 is expressed only in placenta and in the liver in the prenatal period, even when an immunotherapy targeting GPC3 is carried out to suppress tumor growth, adverse events such as autoimmune disease do not occur. This has been confirmed by an experiment using mice.
To date, with regard to glypican-3 (GPC3) as a tumor rejection antigen, the present inventors have identified a peptide, which is mainly presented by HLA-A24 to a killer T cell (International Application No. PCT/JP03/10459; International Filing Date: Aug. 19, 2003). However, with only such peptides presented by HLA-A24 to killer T cells, peptide vaccines can be administered to only the 60% of Japanese people who have HLA-A24. If a peptide presented to a killer T cell by HLA-A2, to which 40% of Japanese people test positive, can be identified, approximately 85% of Japanese people can become the targets of the two types of peptide vaccines. Furthermore, since Caucasians in the Western countries the frequency of HLA-A2 is relatively high, such a peptide presented by HLA-A2 can be applied to many Western people. Accordingly, it is an important object to identify the aforementioned peptide presented by HLA-A2 to a killer T cell. In particular, since melanoma is a cancer, which frequently develops in Caucasians in the Western countries and for which an immunotherapy is effective, and further, since hepatocellular carcinoma has also been rapidly increasing in the Western countries, it is assumed that there would be a large number of patients, for whom an immunotherapy using HLA-A2-binding GPC3 peptide can be applied.